Ceftiofur is a broad-spectrum third generation antibiotic, which is primarily used for veterinary use. It is known chemically as (6R, 7R)-7-[[(2Z)-(2-amino-4-thiazolyl) (methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid and is represented by the formula (I).

Ceftiofur is commercially sold as the sodium salt and is marketed under the brand names Naxcel® and Excenel® for parenteral administration, in bovine animals.
Ceftiofur has been synthesized by any of the following three methods, viz,
I. Ceftiofur and its salts thereof, especially the sodium salt is described in U.S. Pat. No. 4,464,367 (Labeeuw et. al). The patent describes two methods for preparation of Ceftiofur (I) comprising,
    i) amidification at the 7-position of 7-amino-3-thiomethyl furoyl-3-cephem-4-carboxylic acid with a suitably activated [(2Z)-(2-tritylamino-4-thiazolyl) methoxyimino] acetic acid derivative such as mixed anhydride or an activated ester to give ceftiofur (I) after necessary deprotection.    ii) functionalisation at the 3-position of a 7-[2-(2-aminothiazol-4-yl)-2-syn-methoxyimino] acetamido cephalosporanic acid i.e. cefotaxime acid, with thiofuroic acid to give Ceftiofur (I).
U.S. Pat. No. 4,464,367 contains enabling disclosure for preparation of ceftiofur as per method (I) comprising reaction of 7-amino-3-thiomethyl furoyl-3-cephem-4-carboxylic acid with the syn isomer of (2-tritylaminothiazol-4-yl)-2-methoxyimino acetic acid activated with 1-hydroxy benzotriazole, followed by removal of the trityl protecting group to give ceftiofur (I).
However, for method (II), apart from the reaction sequence given in column 3, lines 25-35 depicting conversion of cefotaxime to ceftiofur (I), there is no enabling disclosure whatsoever about how the conversion could be carried out.
The two methods for preparation of ceftiofur of formula (I) are summarized in scheme (I).

Further, synthesis of ceftiofur as per method-I involves additional steps of protection of the amino group of [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid with trityl chloride followed by deprotection in presence of formic acid to give ceftiofur of formula (I). The amidification method utilises toxic, expensive dicyclohexyl carbodiimide for preparing the activated ester of [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid with 1-hydroxy benzotriazole, resulting in the formation of dicyclohexyl urea, which is difficult to remove.
II. Although, the claims of U.S. Pat. No. 6,458,949 B1 (Handa, et al) appear to be obvious, over prior art described in EP Patent No. 0,302,94, U.S. Pat. Nos. 5,109,135, and 5,109,131 the said patent however discloses another method for the preparation of Ceftiofur (I), which is summarized in scheme-II.
The method comprises, reaction of 4-halo-3-oxo-2-methoxyimino butyric acid, activated as the acid halide, with silylated 7-amino-3-thiomethyl furoyl-3-cephem-4-carboxylic acid to give the corresponding 7-carboxamido derivative which on subsequent treatment with thiourea gives ceftiofur of formula (I). Herein, the thiazole ring is formed after the amidification step with thiourea.

The method described in U.S. Pat. No. 6,458,949 B1 is complicated and tedious, as it involves initial preparation of 4-halo-3-oxo-2-methoxyimino butyric acid requiring four steps followed by subsequent amidification reaction with 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid and cyclisation with thiourea. The overall yield of (I) is therefore low, rendering the method commercially not very attractive.
III. U.S. Pat. No. 6,388,070 (Deshpande, et. al) teaches a method for synthesis of ceftiofur (I), and other cephalosporin compounds like ceftriaxone and ceftazidime, cefixime, cefpodoxime acid, cefetamet and cefotaxime acid.
Herein, [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic-acid is activated as the thioester by reaction with 2-mercapto-5-phenyl-1,3,4-oxadiazole in the presence of bis-(2-oxo-oxazidinyl)phosphinic chloride. The thioester on reaction with silylated 7-amino-3-substituted-3-cephem compounds in the presence of a base at low temperatures gives the corresponding 7-acylamido-3-substituted-3-cephem cephalosporin, depicted in scheme-III, herein below:

The activation of [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid with 2-mercapto-5-phenyl-1,3,4-oxadiazole is quite slow and it requires a time upto 34 hours. Moreover, the method utilizes a reagent like bis-(2-oxo-oxazidinyl)phosphinic chloride which is expensive.
Apart from the above methods, which utilize activation of the carboxylic acid moiety of either the [2-(2-aminothiazol-4-yl)]-2-syn methoxyimino acetic acid or 4-halo-3-oxo-2-methoxyimino butyric acid through the formation of a mixed anhydride or activated ester with 1-hydroxy benzotriazole or 2-mercapto-5-phenyl-1,3,4-oxadiazole or an acid chloride, there are no reports of synthesis of ceftiofur (I) by other methods, specially through utilization of other reactive derivatives of the said carboxylic compounds.
Various reactive derivatives of [2-(2-aminothiazol-4-yl)]-2-oxyimino acetic acid compounds have been utilized for synthesis of several 3-substituted cephalosporin antibiotics carrying a [2-(2-aminothiazol-4-yl)]-2-oxyimino acetamido addendum in the 7-position. These include, to a name a few    a) an acyloxypliosphonium halide derivative as disclosed in U.S. Pat. No. 5,317,099 (Lee et. al) for synthesis of cefotaxime and ceftriaxone;    b) an acetyl sulfite dialkyl formiminium halide hydrohalide derivative as disclosed in U.S. Pat. No. 5,037,988 (Meseguer et. al) for synthesis of cefotaxime, ceftriaxone, cefmenoxime, ceftizoxime, and ceftazidime;    c) a dialkyl chloro thiophosphate ester as disclosed in U.S. Pat. No. 5,567,813 (Sung et. al) for synthesis of cefotaxime, ceftriaxone, cefemenoxime, ceftizoxime, cefpirome sulfate and cefepime;    d) a dimethyl forminium chloride chlorosulphate derivative as disclosed in U.S. Pat. No. 5,739,346 (Datta et. al) for synthesis of cefotaxime, ceftriaxone, ceftazidime, cefazolin etc.
A reactive derivative of [(2)-(2-aminothiazol-4-yl)]-2-syn-oxyimino acetic acid compounds widely utilized in cephalosporin chemistry for effecting amidification at 7-position is the 2-benzothiazolyl thioester, first disclosed in U.S. Pat. No. 4,767,852 (Ascher et. al), the chemistry of which is shown hereinbelow.
wherein the groups R1 to R3 are as defined therein and the group R4 is an acetoxy, carbamoyloxy, or is a group of formula S—Y, wherein Y is a heterocyclic ring.
The cephalosporin compounds that fall under the definition of the group R4 of this patent include the commercially and therapeutically valuable cephalosporin antibiotics like cefotaxime, ceftriaxone and cefuzonam.
The group R4 of U.S. Pat. No. 4,767,852 does not encompass ceftiofur as it covers only those 3-thiomethyl compounds in which the sulphur atom is directly attached to a heterocyclic ring, and not those compounds in which a carbonyl group is interposed between S and Y, where Y is a heterocyclic ring Ceftiofur (I) has a carbonyl group interposed between S and Y i.e. between the 3-thiomethyl group and the furan ring.
The method disclosed in U.S. Pat. No. 4,767,852, as is evident from description given in the examples of the said patent, essentially consists reaction of a protected 7-amino-3-substituted cephalosporanic acid derivative, in particular a (N,O)-bis silylated 7-amino-3-substituted cephalosporanic acid derivative with the [2-(2-aminothiazol-4-yl)]-2-syn-oxyimino acetic acid-2-benzothiazolyl thioester in an inert organic solvent at ambient temperature for a time ranging between 0.5 to 48.0 hours to give the object [2-(2-aminothiazol-4-yl)]-2-syn-oxyimino acetamido-3-substituted-3-cephem-4-carboxylic acid compounds such as cefotaxime, ceftriaxone and cefuzonam, which it should be noted carries “a residue of a nucleophile at the 3α-position”.
From the abovementioned methods it is apparent that the preparation of the [2-(2-aminothiazol-4-yl)]-2-oxyimino acetamido-3-substituted-3-cephem4-carboxylic acid compounds is effected essentially in an inert organic solvent, thereby implying a reaction medium free of water and through utilization of a protected 7-amino-3-substituted cephalosporanic acid derivative as the starting material.
In addition, the [2-(2-aminothiazol-4-yl)]-2-oxyimino acetic acid-2-benzothiazolyl thioester has also been utilized for synthesis of other [2-(2-aminothiazol-4-yl)]-2-oxyimino acetamido-3-substituted-3-cephem-4-carboxylic acid compounds such as cefixime as disclosed in U.S. Pat. No. 6,313,289. Here again, the method comprises reacting a protected 7-amino-3-substituted cephalosporanic acid derivative, particularly a protected 7-amino-3-vinyl-3-cephem-4-carboxylic acid, wherein the amino group and the carboxylic group are protected as trialkylsilyl group with a [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolyl thioester in the form of a aqueous solution of a crystalline solvate with dimethyl acetamide and an inert organic solvent medium at room temperature in the presence of a base to give cefixime after removal of the said protective groups.
Cefixime is isolated as the salt of an organic base or as an acid addition salt with sulphuric acid.
In their attempt to extend the method described in U.S. Pat. Nos. 4,767,852 and 6,313,289, for synthesis of ceftiofur the present inventors found to their surprise that when (N,O)-bis silyl-7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III1),
is reacted with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolyl thioester (MAEM) of formula (II),
in an inert organic solvent (dichloromethane) in the presence of an organic base (triethyl amine) at ambient temperature (15-30° C.), the methods had the following disadvantages, which are undesirable for any commercial process. These are,    i) the reaction required a period of more than 20-24 hours to proceed to completion,    ii) the conversion to ceftiofur as indicated by monitoring of the reaction by HPLC was only about 88-90%,    iii) about 9-10% of impurities were formed in the reaction,    iv) the product i.e. ceftiofur was isolated as a gummy material, and    v) the isolated product i.e. ceftiofur had a purity of only about 88% containing about 10-12% of impurites. Purification of the material thus obtained resulted in considerable loss thus giving ceftiofur (I) in low yield.
An improved method for synthesis of ceftriaxone comprising reaction of [2-(2-aminothiazol-4-yl)]-2-syn-methoxyimino acetic acid-2-benzothiazolyl thioester of formula (III) with a unprotected 7-amino-3-substituted cephalosporanic acid derivative i.e. 7-amino-3-(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-1,2,4-triazin-3-yl)thiomethyl-3-cephem-4-carboxylic acid has been disclosed in U.S. Pat. No. 5,026,843 (Riccardo et. al.). The improvement comprises carrying out the said amidification reaction in a monophasic system comprising a mixture of a water-miscible organic solvent and water and in the presence of an organic base. The ceftriaxone thus obtained without isolation is converted into its sodium salt, which is isolated from the reaction mixture. The chemistry is summarized hereinbelow:

The water-miscible organic solvents listed in the U.S. Pat. No. 5,026,843 as useful for carrying out the abovementioned synthesis include tetrahydrofuran, dimethyl acetamide, dimethyl formamide, dioxane, dimethoxyethane. When these solvents are mixed with water, a homogeneous single phase would result. This helps in keeping the ceftiaxone thus produced in solution throughout the reaction and thereby, enabling a one-pot reaction for preparation of ceftriaxone sodium.
An attempt by the present inventors to extend the method described in U.S. Pat. No. 5,026,843 for synthesis of ceftiofur or ceftiofur sodium comprising reaction of [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolyl thioester (MAEM) of formula (II) with 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III),
in a medium consisting of water and a water-miscible organic solvent disclosed in the said patent like tetrahydrofuran and N,N-dimethylacetamide, was however, not satisfactory and was found to give the product i.e. ceftiofur (I) associated with impurities in the level of 5-10% depending on the water-miscible organic solvent used. The product obtained was a sticky solid adhering to the sides of the reaction vessel, rendering its isolation as a solid very difficult.
In addition, replication of the methods exactly as described in U.S. Pat. Nos. 4,464,367 and 6,458,949 B1, referred herein earlier were also found to be associated with formation of higher level of impurities, which are to the tune of about 25-28% and 5-10% respectively.
The level of impurites formed in the-synthesis of ceftiofur by the four methods discussed herein before are summarized in Table-I.
TABLE IThe level of impurities formed in the synthesis of ceftiofur (I) by various methodsTotal%ImpuritiesNo.MethodConversion(%)% YieldPurity %1As per that described in7525-28Gummy material—U.S. Pat. No. 4,464,367(not isolated)2As per that described in91.28.869.692.78U.S. Pat. No. 6,458,949 B13Extrapolation of the method8911Sticky solid—described in U.S. Pat. No. 4,767,852*(not isolated)4Extrapolation of the method93 5-10Sticky solid—described in U.S. Pat. No. 5,026,843**(not isolated)*reaction of (N,O)-bis silyl-7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III1) with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolyl thioester of formula (II) in dichloromethane at ambient temperature**reaction of 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (II) with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid-2-benzothiazolyl thioester of formula (III) in a medium containing water and a water-miscible organic solvent in presence of a base
It might be mentioned herein that the 3-thiofuroylmethyl substituent in ceftiofur, by virtue of it containing a carbonyl group interposed between a sulfur atom and a furan ring system is very labile in nature and is highly susceptible to fission of the sulfur-carbonyl bond as well as highly prone to undergo dimerisation, leading to formation of a dimeric compound in solution. This sets apart ceftiofur (I) from other 3-heterocyclic thiomethyl cephalosporin derivatives as described in U.S. Pat. No. 4,767,852 wherein the abovementioned lability is less pronounced or negligent and thereby ensuring their synthesis through any known methods.
The structure of the impurities arising out of fission of the sulfur-carbonyl bond and dimerization are given herein below as compounds (IV) and (V) respectively.

Thus, to summarize,    i) Synthesis of ceftiofur (I) had been achieved in prior art either through amidification at the 7-amino position of (a suitably protected) 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid activated through formation of a mixed anhydride or through formation of an activated ester with 1-hydroxybenzotriazole, or 2-mercapto-5-phenyl-1,3,4-oxadiazole and described respectively in U.S. Pat. Nos. 4,464,367 and 6,388,070 B1, summarized in Scheme-I and Scheme-III, or through amidification at the 7-amino position of (a suitably protected) 4-halo-2-methoxyimino-3-oxo butyric acid activated as the acid halide, followed by cyclization of the intermediate compound thus formed with thiourea, as described in U.S. Pat. No. 6,458,949 B1 and summarized in Scheme-II;    ii) Functionalization at 3-position of cefotaxime by reaction with thiofuroic acid, as summarized in Scheme-I, for the conversion of which, however, there is no enabling disclosure whatsoever in U.S. Pat. No. 4,464,367;    iii) Replication of both the abovementioned methods was found to give ceftiofur associated with impurities in the range of 5-28%;    iv) Extension of the methods described in U.S. Pat. Nos. 4,767,852 and 5,026,843 for synthesis of ceftiofur (I) was not only lengthy requiring about 18-24 hours but also lead to higher levels of impurity, resulting in a gummy material.    v) The abovementioned methods involve protection and deprotection of reactive functional groups, increasing the cost and time of manufacture;    vi) The product i.e. ceftiofur obtained by the abovementioned methods, because of the higher level of impurities is therefore not suitable for formulation into a suitable dosage form; and    vii) Removal of the impurities by purification leads to considerable loss of the product, increasing the cost of manufacture and rendering such methods commercially not attractive.
Further, since regulatory authorities all over the world are highly concerned about the level of impurities in a drug substance/drug product and are becoming increasingly stringent in approving products containing levels of impurities above the prescribed limits for human or animal consumption, it is imperative that any method of manufacture of a drug substance/drug product, apart from being commercially viable should provide the product conforming to pharmacopoeial specifications, containing amount of impurities within the prescribed limits or subatantially free of such impurities.
In view of the foregoing reasons, there exists a need for a vastly improved method for manufacture of ceftiofur (I), which not only satisfies the techno-commercial aspects i.e. cost-effectiveness, ease of operations, etc, but also provides a product of high purity, free of impurities and possess properties, which are amenable for formulation into a suitable dosage form.
The present inventors have found to their surprise that the existing need for an improved method for manufacture of ceftiofur in high yield and high purity could be achieved through:    i) carrying out amidifaction at 7-amino position of 7-amino-3-thiofuroylmethyl-3-cephalosporanic acid of formula (III) with [2-(2-aminothiazol-4-yl)]-2-methoxyimino acetic acid activated as its -2-benzothiazolyl ester of formula (II), obviating the need for protection and deprotection of reactive functional groups,    ii) the said amidification reaction is carried out in a system comprising mixture of a water-immiscible inert organic solvent and water i.e. a biphasic system and in the presence of a base, in a remarkably shorter time (1.5 to 3.0 hours) resulting in a product with substantially lower level of impurities,    iii) removal of most of the impurities formed during the above reaction in (ii) above, through a selective extraction method to provide ceftiofur (I) of high purity and substantially free of impurities by extraction of the aqueous mixture of the alkyl ammonium salt of ceftiofur (I) with a inert organic solvent.    iv) further removal of impurities formed during acidification of the alkyl ammonium salt of ceftiofur (I) with a mineral acid, in the presence of a mixture of water-miscible and water-immiscible solvent and in the presence of a saturated aqueous solution of an alkali or alkaline earth containing salt, by selectively partitioning ceftiofur (I) in the organic phase substantially free from impurities with the associated impurities getting extracted in the aqueous phase.